Separation and isolation of specific cell types in the context of whole organisms allows their separated cultivation and functional as well as molecular analysis. This has turned out to be very useful for the understanding of specific cell populations and their interaction with other cells but also for their manipulation and therapeutic use.
Numerous methods have been formulated to analyze and sort populations of cells including, but not limited to methods using the size, density or granularity of a cell for a separation by sedimentation which can be performed on its own or in combination with density gradients and centrifugation or elutriation. Further methods are based on the different resistance of cells to osmotic lysis for the separation of e.g. white blood cells from blood. Furthermore, methods of depletion of unwanted cells using specific toxic antibodies reacting with a cell surface marker can be used. Further methods are for example flow cytometry or magnetic cell sorting (e.g. using magnetic bead-conjugated antibodies, e.g. MACS, Miltenyi Biotec) and other methods reliant upon antibody affinity to particular surface molecules like proteins. Using the latter technologies, positive enrichment or depletion of cells can be achieved expressing a certain molecule including but not limited to RNA, DNA, lipid, sugar or protein.
It has been reported that a recombinantly expressed green fluorescent protein (GFP) could be located to the cytosolic face of the plasma membrane of mouse embryonic stem cells due to a palmitoylation site that was present in the recombinant GFP. It was speculated that cells expressing such GFP proteins could be separated from cells not expressing this recombinant GFP using fluorescence activated cell sorting (FACS) (Schindehütte et al. (2005) Stem Cells 23:10-15).
In both flow cytometry and magnetic cell sorting, the protein marker is labeled via a specific antibody which again is directly or indirectly coupled to a fluorescent dye or a superparamagnetic particle (bead). Both, intracellular as well as extracellular markers may be used. However, when living cells need to be obtained, so far only extracellular markers could be used as the cell has to be fixed and permeabilized for intracellular staining.
Therefore, so far only those non transgenic cell types were accessible to magnetic cell sorting and flow cytometry of living cells in the context of a whole organism where surface markers were known and specific, high affinity antibodies were available (Recktenwald and Radbruch, Cell Separation Methods and Applications (1998), 153-174).
As mentioned, many additional cell types are characterized by intracellular markers like cytoskeletal proteins, transcription factors, specific organelle proteins, enzymes, etc. (Berghuis et al. (2004) Int J Dev Neurosci 22:533-43).
Among others, these intracellular markers have been used to generate mouse lines expressing a cell type specific reporter by using the respective promoter (Suzuki et al. (2003) Neurosci Res 47:451-454; Lakso et al. (1992) Proc Natl Acad Sci USA 89: 6232-6236; Zambrowicz et al. (1997) Proc Natl Acad Sci USA 94:3789-94; David et al. (2005) Stem Cells 23:477-482).
Furthermore, certain cell types are characterized in addition or only by spatio-temporal parameters. That means that these cells are not, or not only characterized by the expression of a marker but by their limited occurrence within a certain region or part of an organ or organism and/or by their limited occurrence within a certain time period. For example, cells present only in the amygdalla of the brain, cells having a certain function only during the early postnatal period, cells having certain functions after a lesion of an organ, or cells changing their behavior after a drug treatment.
However, the reporter used in combination with intracellular markers or spatio-temporal properties so far are either fluorescent reporters (like green fluorescent protein (GFP), yellow fluorescent protein (YFP), etc.) or other reporters which can be used to stain a cell (e.g. beta galactosidase). In some cases, transgenes have been introduced which allow for a depletion of a specific cell line by expressing the diphtheria toxin receptor (DTR) (Buch et al. (2005) Nature Methods 2:419-426) or directly the diphtheria toxin A subunit (DT-A) (Palmiter et al. (1987) Cell 50:435-443; Breitmann et al. (1987) Science 238:1563-1565).
Lastly, it has also been reported that under certain conditions and for some markers it may be possible to partially degrade the surface of a cell to access intracellular markers but still keeping a cell alive (Berghuis et al. (2004) Int J Dev Neurosci 22:533-43).
In case of flow sorting, fluorescent reporters can be used for the isolation of living cells. Although high speed flow cytometry sorting instruments have been developed which allow the separation of several ten thousand cells per second, it would be a great step forward if sorting techniques like magnetic cell sorting could be used for the isolation of cells or biological entities derived thereof, characterized by an intracellular marker or spatio-temporal properties. Immunomagnetic cell sorting, e.g. MACS Cell Separation System, allows to separate several billion cells in a few minutes, is much less cumbersome and much more cost effective than flow cytometry, as no complex instrument nor a highly skilled operator are needed.
For cell lines and primary cells which are cultivated ex vivo, several methods have been reported for introduction of a transgenic cell surface marker in order to subsequently make these cells sortable using flow cytometry or magnetic cell sorting (Gaines et al. (1999) Biotechniques 26:683-688). In brief, cells are transfected with a construct leading to the expression of a surface marker which again can be addressed by a specific antibody carrying a fluorescent tag or a superparamagnetic bead. This approach can also be extended to the expression of a transgenic cell surface marker in cells of a whole organism using appropriate transfection methods like viral vectors, pronucleus injection, or gene targeting approaches (Yasunaga et al. (2005) Nat Biotech 23: 1542-1550).
It is, however, not predictable for a person of skill in the art whether a transgenic or transfected whole organism is going to express a reporter as wanted. Exogenous transgenes may not harbor all of the sequences necessary and sufficient for proper regulation of transcription and may therefore be influenced by e.g. cis-regulatory elements near the site of insertion (Banares et al. (2005) Genesis 42:6-16). Therefore, the generation and characterization e.g. of proper transgenic mice is still cumbersome and time consuming.
As for the nature of transgenic surface markers, several different possibilities haven been mentioned in the literature (see above, e.g. CD4, LNGFR, H2Kk, DTR). Most of these markers are naturally occurring proteins which are ectopically expressed to indicate the transgenic status of a cell. Some of these proteins like CD4 and LNGFR have in addition been engineered to have a deleted or mutated intracellular domain in order to avoid signaling upon antibody binding of the extracellular domain. However, none of the proteins mentioned in the literature is adequate to be used in all cell sorting and downstream experiments covering different tissue sources, cell types, and protocols for the application of sorted or sorting cells. Some of these proteins like CD4 are not trypsin or papain resistant and thus cannot be used in protocols in which cells are singularized from solid tissues prior sorting. Some other proteins are multiple transmembrane proteins and are therefore partly not expressed appropriately as a transgene. Also, proteins may be toxic or have at least an unwanted impact on their neighboring cells when expressed ectopically on the cell surface of at least some cell types. Lastly, some proteins, like DTR, are internalized upon antibody reaction.
Furthermore, once cells have been isolated, they are often functionally characterized by grafting them into recipient organisms. For example, the neurogenic potential of neural precursors is assessed by placing them into different brain areas (Seidenpfaden et al. (2006) Mol. Cell. Neurosci. 32:187). Comparably, approaches towards regenerative medicine aim to test different cell types and aggregates derived thereof for their repopulating capacity in model organisms or patients. The rejection of donor cells can in general be avoided by performing autologous transplantations or by using inbred strains in case of preclinical research. But conventional transgenic surface epitopes most probably will trigger an immune response and eventually the rejection of the cells if the recipient is not immune suppressed or the transgenic epitope is not identical to an endogenously expressed epitope. The latter would prohibit distinguishing the grafted cells from the host cells by the transgenic surface epitope after grafting. As will be explained below, this is surprisingly achieved by means of the present invention.